The field of the present invention relates to apparatus and methods for the transfer of macromolecules such as DNA, RNA, proteins and the like from a source medium to a transfer medium, for example, following gel electrophoresis.
The transfer of DNA, RNA or proteins following gel electrophoresis is commonly carried out by three methods. The classical technique is capillary blotting which typically involves placing a piece of transfer membrane in contact with a gel and then placing absorbent paper on the other side of the transfer membrane. The molecules are removed from the gel and transferred to the transfer membrane. Typically in this procedure, the gel is continually re-hydrated by exposing the opposite side of the gel to a thoroughly soaked wick that is in contact with a large reservoir of transfer buffer.
Another method for transferring macromolecules to a transfer membrane involves the application of an electric field across the gel to electrophorese the macromolecules from the gel to the transfer membrane. This method was the first alternative to capillary blotting. It is mainly popular for the transfer of proteins to transfer membranes. However, its popularity was short lived for the transfer of DNA and RNA because of the introduction of a third type of blotting, vacuum blotting.
Vacuum blotting involves the application of a vacuum to one side of the transfer membrane such that fluid is drawn through the transfer membrane from the gel. In this system, the gel is placed in contact with a large buffer chamber directly or with a thoroughly soaked paper wick that is in contact with a large transfer buffer chamber. By application of the vacuum, fluid is drawn out of the gel and through the transfer membrane. This partially dehydrates the gel and the gel must be continually rehydrated by the flow of fluid from the buffer chamber. This flow is typically governed by simple gravitational and capillary forces. U.S. Pat. No. 4,726,889 discloses one such vacuum blotting system.
Of the three methods, vacuum blotting tends to be the most rapid. However, both vacuum blotting and electroblotting have disadvantages, as described below, which tend to limit the effectiveness of those methods, particularly where the macromolecule of interest is DNA or RNA. For that reason, classical capillary blotting is still the most popular method employed for the transfer of DNA and RNA. For protein blotting, however, electroblotting is used almost exclusively.
A disadvantage of electroblotting for DNA and RNA is that the resolution of the DNA fragments on the transfer membrane is not as effective as with capillary blotting nor is the efficiency of the transfer. The disadvantages of vacuum blotting are twofold. First, the technique is somewhat cumbersome and most vacuum blotting apparatus tend to develop leaks of either fluid or air that frequently cause the transfer to be either uniformly inefficient or inefficient in local regions of the transfer membrane. A second disadvantage to vacuum blotting is that only a minimum amount of vacuum can be applied against the transfer membrane and one side of the gel, otherwise the gel will collapse. This is typically a vacuum of about 30-35 mm Hg below atmospheric pressure (all references made hereinafter to pressure are with respect to atmospheric pressure). If a vacuum exceeding about 30-35 mm of mercury is used, fluid is drawn from the gel too rapidly and rehydration of the gel is not quick enough to prevent the gel matrix from collapsing. Collapse of the gel can be recognized by a decrease in the thickness of the gel and results in the cessation of transfer of large molecules from the gel to the membrane. Therefore, once a gel has collapsed the efficiency of the overall transfer is decreased, sometimes substantially. This limitation on the amount of vacuum that can be applied limits both the speed and/or the overall efficiency of the transfer process. Because of the limitations inherent in vacuum blotting, those who work with DNA and RNA have elected to remain with capillary blotting, even though capillary blotting commonly requires six to twelve hours compared to vacuum blotting which requires one to two hours.
In view of the lengthy time periods involved in capillary blotting, the unreliable nature and relative inefficiency of vacuum blotting, and the inaccurate and relatively unfocused transfer of macromolecules using the electrotransfer method, there exists a need for an alternative means to accomplish the transfer of macromolecules to a transfer medium. The present invention meets this need by providing an improved method and apparatus for the transfer of molecular components in an efficient, reliable, and accurate manner.